Biomedical subjects

V Ullrich

Publications and source records attributed to V Ullrich.

At least 37 records · Page 2Linked to original sources

PubMed2000

Effects of the thromboxane receptor agonist U46619 and endothelin-1 on large and small airways.

Recently attention has been drawn to the role of small airways in asthma. However, little information exists about the responsiveness of small airways to various bronchoconstrictors in comparison to large airways. In this study, the model of precision-cut lung slices (PCLSs) was used to investigate the effects of the thromboxane receptor agonist U46619 and endothelin (ET)-1 on small (diameter <250 microm), medium (250-420 microm) and large (>420 microm) airways. Viable PCLSs were prepared from rat lungs and the bronchoconstriction of differently sized airways inducible by U46619 and ET-1 was observed by means of a microscope and analysed by digital imaging techniques. The median effective concentration (EC50) of U46619 for inducing bronchoconstriction was 6.9 nM in small and 66 nM in large airways, respectively. This finding was corroborated by direct observations in single lung slices containing both a small and a large airway. In such slices, U46619 caused smaller airways to contract to a greater degree than larger ones. ET-1 induced bronchoconstriction was similar in small (EC50 34 nM) and in medium or large (ECso 22 nM) airways. This was again confirmed by direct observation of ET-1-treated PCLSs. It is concluded that, in rat lungs, endothelin-1 affects small and large airways to the same extent, whereas thromboxane is ten times more potent in causing small airways to contract than larger ones. Precision-cut lung slices appear to be a valuable model for examining the (patho)physiology of small airways.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2000

Nitration and inactivation of cytochrome P450BM-3 by peroxynitrite. Stopped-flow measurements prove ferryl intermediates.

Peroxynitrite (PN) is likely to be generated in vivo from nitric oxide and superoxide. We have previously shown that prostacyclin synthase, a heme-thiolate enzyme essential for regulation of vascular tone, is nitrated and inactivated by submicromolar concentrations of PN [Zou, M.-H. & Ullrich, V. (1996) FEBS Lett. 382, 101-104] and we have studied the effect of heme proteins on the PN-mediated nitration of phenolic compounds in model systems [Mehl, M., Daiber, A. & Ullrich, V. (1999) Nitric Oxide: Biol. Chem. 2, 259-269]. In the present work we show that bolus additions of PN or PN-generating systems, such as SIN-1, can induce the nitration of P450BM-3 (wild-type and F87Y variant), for which we suggest an autocatalytic mechanism. HPLC and MS-analysis revealed that the wild-type protein is selectively nitrated at Y334, which was found at the entrance of a water channel connected to the active site iron center. In the F87Y variant, Y87, which is directly located at the active site, was nitrated in addition to Y334. According to Western blots stained with a nitrotyrosine antibody, this nitration started at 0.5 microM of PN and was half-maximal between 100 and 150 microM of PN. Furthermore, PN caused inactivation of the P450BM-3 monooxygenase as well as the reductase activity with an IC50 value of 2-3 microM. As two thiol residues/protein molecule were oxidized by PN and the inactivation was prevented by GSH or dithiothreitol, but not by uric acid (a powerful inhibitor of the nitration), our data strongly indicate that the inactivation is due to thiol oxidation at the reductase domain rather then to nitration of Y residues. Stopped-flow data presented here support our previous hypothesis that ferryl-species are involved as intermediates during the reactions of P450 enzymes with PN.

Animals↗

PubMed2000

Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxide. Evidence for a dithiol-disulfide equilibrium and implications for redox regulation.

Calcineurin (CaN) is a Ca2+-and calmodulin (CaM)-dependent serine/threonine phosphatase containing a dinuclear Fe-Zn center in the active site. Recent studies have indicated that CaN is a possible candidate for redox regulation. The inactivation of bovine brain CaN and of the catalytic CaN A-subunit from Dictyostelium by the vicinal dithiol reagents phenylarsine oxide (PAO) and melarsen oxide (MEL) and by H2O2 was investigated. PAO and MEL inhibited CaN with an IC50 of 3-8 microM and the inactivation was reversed by 2, 3-dimercapto-1-propane sulfonic acid. The treatment of isolated CaN with hydrogen peroxide resulted in a concentration-dependent inactivation. Analysis of the free thiol content performed on the H2O2 inactivated enzyme demonstrated that only two or three of the 14 Cys residues in CaN are modified. The inactivation of CaN by H2O2 could be reversed with 1,4-dithiothreitol and with the dithiol oxidoreductase thioredoxin. We propose that a bridging of two closely spaced Cys residues in the catalytic CaN A-subunit by PAO/MEL or the oxidative formation of a disulfide bridge by H2O2 involving the same Cys residues causes the inactivation. Our data implicate a possible involvement of thioredoxin in the redox control of CaN activity under physiological conditions. The low temperature EPR spectrum of the native enzyme was consistent with a Fe3+-Zn2+ dinuclear centre. Upon H2O2-mediated inactivation of the enzyme no significant changes in the EPR spectrum were observed ruling out that Fe2+ is present in the active enzyme and that the dinuclear metal centre is the target for the oxidative inactivation of CaN.

Animals↗

PubMed1999

Prostacyclin synthase is localized in rat, bovine and human neuronal brain cells.

Using a new polyclonal antibody against prostacyclin (PGI2)-synthase this enzyme was shown to be present in neuronal cells of bovine, rat and human brain, most abundantly in Purkinje cells of the cerebellum and cortical neurons, but not in glial cells. Western blots confirmed the specificity of the antibody and applied to enriched neuronal and astrocyte cultures supported these immunohistochemical data. It was further shown that staining with an anti-nitrotyrosine antibody was positive for PGI2-synthase containing cells. Possible physiological and/or pathophysiological functions of the enzyme in brain are discussed.

Aged↗

PubMed1999

Peroxynitrite reaction with heme proteins.

We have reported that low levels of peroxynitrite (PN) can cause inactivation of the heme-thiolate protein prostacyclin (PGI2)-synthase by nitration of a tyrosine residue. To prove that iron catalysis is involved we studied the interaction of PN with microperoxidase and P450nor, a heme-thiolate protein of known structure. Spectral and kinetic analyses allow to conclude on a ferryl nitrogen dioxide complex as an intermediate which decomposes in the presence of an excess of PN under formation of dioxygen, nitrite, and nitrate. This occurs in a catalytic cycle which was more efficient with P450nor than with microperoxidase. If phenol was added to the reaction mixtures of PN and the ferric complexes the ratio of hydroxylated to nitrated phenols decreased compared to the metal-free system. Phenol competed with the formation of dioxygen indicating that the ferryl intermediate was involved in both pathways. One therefore can postulate that the ferryl complex reacts with phenol to give the phenoxyradical which is nitrated in the presence of nitrogen dioxide but does not give hydroxylated products as with metal-free PN. Alternately, the ferryl nitrogen dioxide complex can oxidize a second PN molecule to the radical, *OONO, which can decompose to dioxygen and NO. The latter forms N2O3, with the remaining *NO2 radical. A third pathway consists in the isomerization to nitrate which also is catalyzed by the heme proteins since the ratio of nitrite/nitrate does not change significantly during the catalytic reaction with excess of PN. Our data explain the mechanism of nitration of PGI2-synthase, suggest a role of P450nor as a PN scavenger, and favor heme-thiolate complexes for trapping PN.

Catalysis↗

PubMed1999

Selective nitration of prostacyclin synthase and defective vasorelaxation in atherosclerotic bovine coronary arteries.

Prostacyclin synthase (PCS) is an enzyme with antithrombotic, antiproliferative, and dilatory functions in the normal vasculature, and inactivation of PCS by tyrosine nitration may favor atherosclerotic processes. Here, we show that PCS is nitrated and inactivated in early stage atherosclerotic lesions (focal intimal thickenings). Immunoprecipitation with antibodies raised against nitrotyrosine yielded PCS as the main nitrated protein in blood vessels. Moreover, we identified two nitrated degradation products of PCS with molecular mass of 30 and 46 kd, which were selective for atherosclerotic tissue. Agonist (acetylcholine, angiotensin II)-induced prostacyclin formation was decreased in atherosclerotic vessels compared with normal tissue, whereas PGE2 formation was increased and cyclooxygenase activity remained unchanged. A selective loss of PCS activity was confirmed by direct measurement of enzymatic activity. In line with this, we observed defective relaxation of early atherosclerotic vessels following vasoconstrictive stimulation. This functional impairment was completely reversed by coincubation with an antagonist of the thromboxane/PGH2 receptor but not by a thromboxane synthase inhibitor. These data suggest that reduced PCS activity in atherosclerotic arteries prevents the rapid use of PGH2, which accumulates and acts as an agonist on the vasoconstrictive thromboxane receptor.

Animals↗

PubMed1999

Effects of suramin on PMN interactions with different surfaces.

Human polymorphonuclear leukocytes (PMN) were found to tightly adhere on endothelial (lines EAhy926 and ECV304) and collagen surfaces under the influence of the chemotherapeutic drug suramin. This was observed by scanning electron microscopy and quantitated by myeloperoxidase assays. Suramin also inhibited Ca2+ ionophore A23187-stimulated leukotriene (LT) synthesis in PMN interaction with endothelial cells or with collagen surface. Suramin decreased the release of radiolabeled arachidonic acid (AA) and 5-lipoxygenase (5-LO) metabolites by prelabeled PMN stimulated with A23187. Using agents releasing the suramin-stimulated adhesion namely jasplakonolide and dextran sulfate, we observed a reversal of the suramin effect on leukotriene synthesis. Jasplakonolide released the adhesion of PMN on endothelial and collagen-coated surfaces and restored 5-LO activity. Dextran-sulfate released adhesion on collagen-coated surfaces and abolished suramin inhibition. Arachidonate could also overcome adhesion and inhibition of 5-LO. We conclude that suramin-induced tight attachment of PMN on to solid surfaces lead to decreased leukotriene synthesis during subsequent A23187 stimulation in the absence of exogenous substrates.

Arachidonate 5-Lipoxygenase↗

PubMed1999

Prostaglandin endoperoxide-dependent vasospasm in bovine coronary arteries after nitration of prostacyclin synthase.

In the present study we used a bioassay to study the effects of peroxynitrite (ONOO-) on angiotensin II (A-II)-triggered tension in isolated bovine coronary arteries in order to show the consequences of the previously reported PGI2-synthase inhibition by ONOO- in this model. The following results were obtained: 1. 1 micromol L(-1) ONOO- impaired A-II-induced vasorelaxation and caused a second long lasting constriction phase. Indomethacin (10(-5)M) prevented both effects. U51605, a dual blocker of PGI2-synthase and thromboxane (TX)A2-synthase mimicked the effects of ONOO-. 2. The selective TXA2/prostaglandin endoperoxide (PGH2) receptor antagonist SQ29548 antagonized the second vasoconstriction phase after ONOO- -treatment. Since a generation of TXA2 and 8-iso-prostaglandin F2alpha could be excluded a direct action of unmetabolized PGH2 on the TXA2/PGH2 receptor was postulated. 3. ONOO- dose-dependently inhibited the conversion of 14C-PGH2 into 6-keto-PGF1alpha in isolated bovine coronary arteries with an IC50-value of 100 nM. 4. Immunoprecipitation of 3-nitrotyrosine-containing proteins with a monoclonal antibody revealed PGI2-synthase as the only nitrated protein in bovine coronary arteries treated with 1 micromol 1(-1) ONOO-. 5. Using immunohistochemistry a co-localization of PGI2-synthase and nitrotyrosine-containing proteins was clearly visible in both endothelial and vascular smooth muscle cells. We concluded that ONOO- not only eliminated the vasodilatory, growth-inhibiting, antithrombotic and antiadhesive effects of PGI2 but also allowed and promoted an action of the potent vasoconstrictor, prothrombotic agent, growth promoter, and leukocyte adherer, PGH2.

Angiotensin II↗

PubMed1999

Peroxynitrite inactivates prostacyclin synthase by heme-thiolate-catalyzed tyrosine nitration.

Previous work has shown a sensitive inhibition of prostacyclin synthase activity by peroxynitrite as well as by superoxide in the presence of NO donors. Neither superoxide nor NO alone nor decomposed peroxynitrite is effective. The inhibition of activity was paralleled by a nitration of a tyrosine residue and both could be prevented by a stable substrate analog. The same IC50 value for peroxynitrite was also found for the cellular prostacyclin activity in endothelial and kidney mesangial cells, indicating that the antioxidant potential of the cell cannot prevent the inactivation. Aortic tissue shows a co-localization of prostacyclin synthase and nitrotyrosine staining after treatment of the tissue with 1 microM peroxynitrite. It can be speculated that this pathway of enzyme nitration is of pathophysiological significance.

Animals↗

PubMed1998

Interleukin 1beta decreases prostacyclin synthase activity in rat mesangial cells via endogenous peroxynitrite formation.

We have reported that peroxynitrite (PON) selectively inactivated prostacyclin synthase (PGIS) by a mechanism of tyrosine nitration at the active site [Zou, Martin and Ullrich (1997) Biol. Chem. Hoppe-Seyler 378, 707-713]. We have now extended our studies on rat mesangial cells (RMC) and show that nitration can occur under the influence of cytokines. Pretreatment of RMC with interleukin 1beta (IL-1beta), which up-regulated cyclo-oxygenase 2 and inducible nitric oxide synthase (NOS-2), significantly attenuated the conversion of [14C]prostaglandin H2 (PGH2) into the stable prostacyclin (PGI2) metabolite 6-oxo-prostaglandin F1alpha (6-oxo-PGF1alpha). The presence of superoxide dismutase (SOD, 100 units/ml) or the NOS synthase inhibitor Nomega-monomethyl-l-arginine (100 microM) as well as cycloheximide (10 microM) plus actinomycin (10 microM) abolished IL-1beta-mediated down-regulation of 6-oxo-PGF1alpha from PGH2. At the same time, 6-oxo-PGF1alpha production from arachidonate (AA) increased at the expense of prostaglandin E2 (PGE2). Neither NO alone generated from different NO donors nor superoxide from xanthine/xanthine oxidase (1-100 m-units/ml) inhibited PGI2 synthesis, either from PGH2 or from AA. Bolus additions of chemically synthesized PON or the PON generator 3-morpholinosydnonimine N-ethylcarbamide (SIN-1) exhibited a potent inhibition of 6-oxo-PGF1alpha release from both PGH2 and AA. In addition, immunoprecipitation of nitrotyrosine-containing proteins from PON- and SIN-1-treated RMC yielded distinct nitrated PGIS bands but also from IL-1beta-pretreated cells alone, compared with a lack of nitrated PGIS in control cells. Taken together, our results strongly suggest that IL-1beta pretreatment of RMC via NOS-2 leads to the production of PON with the consequence of a partial nitration and inhibition of PGIS.

Animals↗

PubMed1998

Involvement of ecto-ATPase and extracellular ATP in polymorphonuclear granulocyte-endothelial interactions.

The adhesion of human polymorphonuclear granulocytes (PMN) with confluent human endothelial cells (line EAhy926) and with solid substrate coated by collagen and fibronectin (Fn) was studied by phase contrast microscopy and by the measurement of myeloperoxidase activity. The ecto-ATPase inhibitors suramin and Reactive Blue 2 (RB2) more than doubled the adhesion of PMN to endothelial cells. The cells hydrolyzed added ATP and this reaction was inhibited by suramin and RB2. The degree of ATP hydrolysis during PMN adherence depended on solid substrata and decreased in the order: non-stimulated endothelial cells, TNF-stimulated endothelial cells, collagen-coated surface, Fn-coated surface. In the same order adherence increased. The endogenous level of extracellular ATP in the PMN-endothelial coculture was around 25 nM. We conclude that PMN-endothelial adhesion is counteracted by an ecto-ATPase or by ATP receptors with ATPase activity. Such interactions may play a role in PMN rolling and diapedesis as well as in the pathophysiology of PMN activation by an anergic endothelium.

Adenosine Triphosphatases↗

PubMed1998

New aspects in the reaction mechanism of phenol with peroxynitrite: the role of phenoxy radicals.

The decomposition of peroxynitrite (PON) in aqueous solutions was investigated by monitoring the release of dioxygen as a function of pH together with the various reaction products generated from phenol. This substrate was used as a mechanistic model for tyrosine nitration in prostacyclin synthase for which we have reported a highly efficient nitration and inhibition by PON (Zou, M., Martin, C., and Ullrich, V. (1997) Biol Chem. 378, 707-713). Nitrite as a contaminant and product of PON generated 4-nitrosophenol and some nitrophenols in the acidic pH range. In the alkaline range high amounts of 4-nitrosophenol originated from the disproportionation of PON yielding dioxygen and NOx species. The hydroxylation of phenol occurred between pH 3 and 8 with a maximum at 4.5. The nitration by PON also required a pH between 4 and 8 but had a second maximum between 10 and 12, suggesting that in this pH range phenolate was the reacting species. All isomeric biphenols were found as dimerization products as well as 4-phenoxyphenol (4-hydroxydiphenyl ether), indicating phenoxy radicals as intermediates. Since anisol when incubated under the same conditions yielded only hydroxylation but virtually no nitration products, it was concluded that nitration of phenolic compounds requires a one-electron oxidation as a primary step, followed by addition of the nitrogen dioxide radical.

Anisoles↗

PubMed1998

On the induction of cyclooxygenase-2, inducible nitric oxide synthase and soluble phospholipase A2 in rat mesangial cells by a nonsteroidal anti-inflammatory drug: the role of cyclic AMP.

One of the challenges in the therapy with anti-inflammatory drugs is the avoidance of gastrointestinal side effects, which may be achieved by selective inhibition of cyclooxygenase (COX) -2. CGP 28238 is reported with these characteristics inhibiting selectively the COX-2 activity at nanomolar concentrations. However, we report here on a novel action of this compound uncovered during the application of higher concentrations. In rat mesangial cells, CGP 28238 induced the mRNA and the protein of COX-2 as well as those of inducible nitric oxide synthase and soluble phospholipase A2. In the case of COX-2, this stimulation had no effect on the production of COX-2 metabolites because of the effective blockade of the enzyme. In contrast, the level of NO produced by the cells increased in a concentration-dependent manner from 1.2 to 12.5 nmol of nitrite/3 x 10(5) cells. Furthermore, in combination with low doses of IL-1 CGP 28238 superinduced the formation of nitrite. The observed effects were independent of the inhibition of prostaglandin formation, as suggested by the failure of the potent COX inhibitor diclofenac to cause similar effects. Furthermore, the activity and expression of enzymes downstream of the COX step, such as prostacyclin synthase, were unaffected by CGP 28238. The inductive action of CGP 28238 could be blocked by inhibitors for tyrosine kinases and protein kinase A, such as genistein and KT5720, respectively. The increase in intracellular cAMP concentration in rat mesangial cells and the inhibition by CGP 28238 of phosphodiesterase 4 activity with an IC50 value of 23 muM gave a rationale to explain the underlying mechanisms for the induction of the inflammatory response genes COX-2, soluble phospholipase A2 and inducible NO synthase in rat mesangial cells.

Animals↗

PubMed1998

Dependence of neutrophil activation on cell density and adhesion.

Upon an increasing cell density human neutrophils develop more cell-to-cell contacts in conjunction with an increase in the pHi. These changes are accompanied by decreased superoxide formation after adherence, and a decrease in the total amount of 5-lipoxygenase products after various stimuli. Among the various arachidonate metabolites, leukotriene formation remained almost constant but the yield in 5-HETE decreased. This drop in could account for the decrease in total 5-lipoxygenase products observed when the cell density increased. We conclude that cellular signalling can be affected by an increase of cell-cell interactions. Whether the increase in cellular pH is a cause or consequence of such contact inhibition has yet be answered.

Arachidonate 5-Lipoxygenase↗

PubMed1997

Properties of prostacyclin synthase.

Prostacyclin synthase (PGIS) was isolated from bovine aortic microsomes after detergent solubilisation following purification by DEAE-Sephacel, immobilized metal affinity, and hydroxy apatite chromatography. The homogenous protein exhibited spectral characteristics of a heme-thiolate protein (P450) like the enzyme purified earlier from porcine microsomes and had an apparent mass of 52 kDa on SDS/PAGE. Three peptides from an endoproteinase Lys-C digest were isolated and sequenced. An antiserum was prepared from rabbits and purified by affinity chromatography. This allowed Western blots of microsomes from cultured endothelial cells. After treatment with IL-1 the activity of the cells in producing 6-keto-PGF1 alpha increased about threefold over 27 h which was accompanied by an increase in PGIS mass. A monoclonal antibody was used to set up an ELISA which served for the quantitation of PGIS in bovine tissues.

Amino Acid Sequence↗

PubMed1997

Wortmannin interferes with thrombin-evoked secondary calcium redistribution in human platelets.

Wortmannin has previously been reported to inhibit calcium entry in thrombin-stimulated human platelets. We extend these findings by demonstrating that the redistribution of calcium from intracellular stores features two separate, consecutive phases the second of which is selectively abolished by wortmannin. The primary release of calcium from Ins 1,4,5 P3-sensitive stores remains unaffected. Hence, wortmannin is interfering with regulation of any secondary, sustained calcium accumulation in the cytosolic compartment of activated platelets, originating either from intracellular stores or from calcium entry. We assume that wortmannin blocks a common step in receptor-dependent regulation of calcium entry. We assume that wortmannin blocks a common step in receptor-dependent regulation of calcium entry and intracellular calcium circulation.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1997

Tyrosine nitration as a mechanism of selective inactivation of prostacyclin synthase by peroxynitrite.

Vascular tone critically depends on the endothelial release of nitric oxide and prostacyclin. Superoxide anions counteract these relaxations by trapping nitric oxide under formation of peroxynitrite. As we have recently reported, peroxynitrite is able to inhibit prostacyclin formation in aortic microsomes (Zou et al., 1996). Here we show that peroxynitrite also blocks purified prostacyclin synthase with an IC50 value of about 50 nM and with a similar sensitivity also inhibits the enzyme activity in the EaHy 926 endothelial cell line. Thromboxane synthase, having the same heme-thiolate (P450) structure and a closely-related mechanism was unaffected by peroxynitrite. Anti-nitrotyrosine antibodies reacted positive by a Western blot after treatment of the purified enzyme with 1 microM peroxynitrite. Tetranitromethane also inhibited the enzyme activity which, like the inhibition by peroxynitrite, could be partially prevented in the presence of the substrate analog U46619. The simultaneous generation of superoxide and nitric oxide proved to be as efficient as a bolus of peroxynitrite which supports a possible inactivation of prostacyclin synthase under in vivo conditions. This substantiates an often suggested crucial role of superoxide in the pathophysiology of the cardiovascular system.

Animals↗

PubMed1996

Ebselen-binding equilibria between plasma and target proteins.

The antiinflammatory drug ebselen (2-phenyl-1,2-benzisoselenazo-3(2H)-one) is known to bind covalently to thiols to form seleno disulfides that, directly or indirectly, are responsible for its pharmacological effects. Due to its reactive thiol group and high plasma concentration, albumin is a preferred target of ebselen, which it binds covalently. Ebselen should not, then, be available for intracellular actions at other target proteins. We have addressed this question, and show by difference spectroscopy that the interaction of ebselen with albumin occurs stoichiometrically under ring opening, but is readily reversible in the presence of glutathione. With intact human polymorphonuclear leukocytes (PMN), a similar stoichiometric reaction with distinct spectral features was observed with ebselen that was completely abolished by pretreatment of PMN with N-ethylmaleimide, but not by selective depletion of cellular glutathione. Human platelets, again, exhibited different spectral changes upon addition of ebselen. In agreement with results reported in the literature, we show that 14C-ebselen is in dynamic equilibrium with all accessible thiol groups and, hence, despite mostly being bound covalently to albumin, it will exchange rapidly with other target proteins in PMN or platelets.

Anti-Inflammatory Agents, Non-Steroidal↗